We and others have previously shown the successful use of gadolinium labeled Aβ peptides to target amyloid plaques in transgenic AD model mice [26
]. This approach for labeling amyloid lesions takes advantage of the high affinity binding Aβ peptides have to other Aβ peptides [18
]. However, Aβ peptides are toxic and deposited Aβ can seed further amyloid deposition [18
]. It has previously been shown that circulating Aβ peptides can cross the BBB in vivo and contribute directly to amyloid lesion growth [23
]. Therefore in the present study we sought to develop Aβ homologous peptides as amyloid targeting agents, which are non-toxic and non-fibrillogenic. We designed these homologous peptides so that they still have a high binding affinity to wild-type Aβ peptides and also have a similar BBB permeability. In prior studies we have shown that these Aβ homologous peptides do not self-assemble or promote the fibrillization of endogenous Aβ peptides [2
]. In the current study we used K6-Aβ1–30 chelated to gadolinium via incorporation of DPTA at the amino terminus. In our previously published studies we used K6-Aβ1–30 as a vaccine therapy in AD model mice and have shown that this peptide is both non-toxic and non-fibrillogenic [35
]. In the current study we have also demonstrated that this Aβ homologous peptide is non-toxic when labeled with chelated Gd (). Importantly, we show that Gd-DTPA-K6Aβ1–30 maintains its high affinity binding to Aβ1–42, allowing its use as an amyloid targeting agent. As we have previously reported using Aβ1–40, the introduction of Gd-DTPA lowers BBB permeability significantly [42
]. Hence, in order to use this ligand for amyloid labeling the BBB has to be transiently disrupted with mannitol. In this study we document that Gd-DPTA-K6Aβ1–30 is able to label amyloid lesions in vivo when the BBB is disrupted with mannitol.
Similar to previous ex vivo- [22
] and in vivo reports [16
], we were able to image some amyloid lesions in our AD transgenic mice without the use of a contrast agent. With our relatively short imaging times, only a small proportion of amyloid lesions could be detected without the use of a contrast agent. This direct detection of amyloid lesions most likely reflects iron content within plaques [6
]. Iron deposition has been previously reported both in AD and in transgenic AD mouse model plaques, in particular in more mature lesions [38
]. We detected plaques without contrast agent mainly in older animals and in large plaques, most likely related to the increased iron content within some of these more mature lesions. However, when we performed voxel based analysis comparing the pre-ligand MRI scans of controls versus the Tg AD model mice, there were no significant differences, reflecting the small percentage of lesions which can be detected without contrast agent. Detection of AD pathology is most important at the earliest stages of disease, since that is the point at which therapeutic interventions, which are currently under development, will have their greatest effect [45
]. For the identification of these earlier lesions contrast agents will likely be needed. Other promising approaches under development include MRI detection of 19F-labelled amyloidophilic compounds as reported in transgenic mice [13
]. Significantly, our MRI based methods are able to detect amyloid deposits in transgenic mouse models of AD even when the amyloid burden is relatively low. PET based methods, such as those using PIB are unable to detect plaques in mice even when their amyloid burden is high as in 12 month old PS1/APP mice [21
]. Hence, for studies developing amyloid clearing agents where transgenic models are being used to evaluate efficacy, MRI approaches are preferred.
In this study we report the first use of a voxel based analysis (VBA) of AD model μMRI. Statistical Parametric Mapping [SPM’99, Wellcome Department of Cognitive Neurology, London, UK] [8
] is a collection of tools available in the public domain for basic visualization and analysis of brain images. It is routinely applied for the analysis of structural and functional brain images in humans and it has been recently applied to an autoradiographic study in rats [25
]. SPM is predominantly used for its convenience in statistical examination of group differences. VBA, as performed with SPM, relies on semi-automated image registration, spatial normalization and smoothing procedures to standardize all brains to a common space and allow assessment of the brain images on a voxel-wise basis. With registration and normalization, one assumes that all structures occupy the same volume and have the same shape. During VBA facilitates examination of large data sets through the rapid creation of statistical maps that enable to localize significant changes in the whole brain and on a voxel-wise basis. This represents a powerful, unbiased tool to assess the potential effectiveness of therapeutic interventions for amyloid removal, many of which are currently being developed using AD model mice. In the present study VBA comparison of pre and post-ligand injection allowed us to definitively identify ligand binding associated with amyloid. In our studies the pre-ligand and post-ligand injection scans were done 2 weeks apart; over such a short interval it is very unlikely significant changes in the volume of brain structural will have occurred. However, age related volume changes in the brain structures in AD model and wild-type mice do occur and could confound comparisons between scans done too far apart temporally [29
]. With our imaging protocol, it was important to have pre-ligand injection scans so that dark spots that are associated with vessels (or other dark structures) rather than plaques can be differentiated. Binding of our ligand to amyloid lesions results in increased darkness on the T2* imaging sequences in areas of plaques. Without comparison to pre-ligand injection scans (or amyloid immunohistochemical staining on tissue sections) it is not possible to definitively identify dark spots as plaques. Hence, application of our ligand and imaging protocols for amyloid detection need to be combined with VBA for making a definitive distinction between mice with amyloid plaques and control non-transgenic mice. As shown in , when our ligand was injected into control mice, no significant difference was noted between pre and post ligand injection scans, contrasting with our findings when our ligand was injected into AD transgenic mice. Hence, our ligand is associated with little or no non-specific signal change on T2* imaging in mice without amyloid lesions.
In the present study we have not directly compared the sensitivity and specificity of VBA versus a region-of-interest (ROI) approach for quantitation of differences in the amyloid burden. It is possible that an ROI approach may have greater sensitivity; however, we focused on VBA as this method is accurate and much more rapid to apply [24
A limitation of our imaging protocol is the need to open the BBB with the use of mannitol, along with carotid artery clamping during the injection of the ligand. The use of modifications of gadolinium labeled Aβ1–40, such as putrescine conjugation, to increase the BBB permeability have been reported [19
]; however, when we performed putrescine modification of Gd-K6Aβ1–30 we could not obtain consistent labeling of amyloid deposits with this ligand (data not shown). Several other modifications of ligands to increase their BBB permeability have been reported such as incorporation of poly-cationic domains and coupling with proteins that are actively transported into the brain [7
]. We are currently investigating such methods to increase the BBB permeability of our ligand. It is only with overcoming this problem of BBB permeability that our ligand would have the possibility of being applied to humans.
Our present findings support the use of a contrast imaging agent for early plaque detection, using non-toxic, non-fibrillogenic Aβ derivatives such as K6Aβ1–30. Such an amyloid contrast probe enhances the sensitivity of plaque detection that allows following the amyloid burden in individual AD mice longitudinally. This will greatly aid the development of therapeutic agents which aim to remove existing amyloid plaques.